PROJECT SUMMARY AMPA-subtype ionotropic glutamate receptors, which mediate fast signaling between neurons and contribute to high cognitive processes, are also implicated in numerous neurological diseases, including Alzheimer's disease, amyotropic lateral sclerosis, epilepsy and ischemia. The ability to regulate AMPA receptors is therefore an important clinical goal. There is, however, an unmet need for drugs to regulate AMPA receptor activity in pathological conditions that indicates a profound gap in our knowledge of AMPA receptor structure and function. Our long-term goal is to understand how AMPA receptor molecular machinery works at the atomic level. We plan to study AMPA receptor structure and function using advances in single-particle cryo- electron microscopy (cryo-EM). Not only can modern cryo-EM reach similar or better resolution than X-ray crystallography, it can also be used as a powerful tool to analyze heterogeneous populations of AMPA receptor particles and extract structural information that belongs to each individual functional conformation. Because of much more economic sample requirements, cryo-EM can also approach complexes of AMPA receptor with transmembrane regulatory subunits, which are hard to produce in large quantities and demonstrate high degree of conformational and stoichiometric heterogeneity and are thus intractable to X-ray crystallography. Our Specific Aims are to (1) elucidate structural basis of AMPA receptor regulation by auxiliary subunits, (2) determine molecular bases of AMPA receptor activation, and (3) establish molecular mechanism of AMPA receptor desensitization. To reach our goals, we will use cryo-EM to obtain structures of rat GluA2 in complex with different auxiliary subunits in the presence of competitive antagonists, agonists and/or positive allosteric modulators. We will use Fluorescence-detection Size Exclusion Chromatography (FSEC) and thermostability assays to assess protein expression, assembly, homogeneity and stability. We will also employ site-directed mutagenesis and electrophysiological recordings to study functional mechanisms and to critically test our structural models. Reaching our research goals will have a significant impact on understanding the mechanisms of excitatory neurotransmission and will provide molecular-level knowledge essential to facilitate design of new drugs to treat neurological diseases.